Natural killer cell-specific antigen and antibodies that identify the same

ABSTRACT

A novel natural killer cell-specific molecule, designated as PEN5, consisting essentially of a glycoprotein pair called PEN 5 α and PEN5β, having apparent molecular weights of 120-150 and 210-245 kdal, respectively. Monoclonal antibodies, including immunoreactive fragments and derivatives thereof, that bind to unique epitopes present on this NK, cell-specific molecule; hybridomas that produce the monoclonal antibodies; methods of using the antibodies and fragments and derivatives; and methods for detecting and/or removing natural killer cells from a sample containing a mixed population of cells are also provided.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional application of Ser. No.08/484,748 filed Jun. 7, 1995 now issued as U.S. Pat. No. 5,786,160 onJul. 28, 1998, which is a continuation-in-part of Ser. No. 08/113,170filed Aug. 27, 1993, now abandoned; and is a continuation ofPCT/US94/09714 filed Aug. 26, 1994, now in the national stage.

STATEMENT OF GOVERNMENT RIGHTS IN INVENTION

This work was supported by a National Institutes of Health grant,CA53595. The government of the United States of America has certainrights to this invention.

FIELD OF THE INVENTION

The invention relates to novel cell surface structures that areselectively expressed on a subpopulation of natural killer cells and toantibodies that bind to unique epitopes on these structures.

BACKGROUND OF THE INVENTION

Natural killer cells (hereinafter sometimes referred to as “NK cells”)are large granular lymphocytes (“LGLs”) comprising 2-15% of peripheralblood mononuclear cells in healthy individuals. Although NK cells do notrearrange or express either of the known T cell receptor complexes, theycan recognize and kill certain virus-infected and transformed cells in anon-MHC-restricted fashion, without prior sensitization. With theexception of CD16, an Fc receptor for immunoglobulin that recognizesantibody-coated target cells, the NK cell surface receptors responsiblefor target cell recognition have not been identified. The lack of adefining surface receptor requires NK cells to be identified by acombination of phenotypic and functional characteristics.

Although most NE cells are CD3:TCR−, CD16+, CD56+ LGLs, there isconsiderable phenotypic and functional heterogeneity within thispopulation (Trinchieri, Adv. Immunol., 47:187 (1989)). For example, thesurface density of CD56 has been shown to define functionally distinctNK cell populations. CD56 ^(bright) NK cells are largely CD16 ⁺,agranular lymphocytes deficient in cytolytic effector function thatproliferate vigorously in response to exogenous IL-2. CD56 ^(dim) NKcells are CD16 ⁺ LGLs possessing potent cytolytic effector function thatdo not proliferate in response to IL-2. Because some T cells expressboth CD16 and CD56, these molecules, by themselves, cannot define the NKcell population. (Trinchieri, 1989). Furthermore, because the expressionof CD56 on the functionally differentiated population of NK cells islow, monoclonal antibodies reactive with CD56 cannot be used to reliablydistinguish this subpopulation of NK cells from other cells in a sample.

It is an object of the present invention to identify a novel cellsurface structure that is preferentially expressed on functionallydifferentiated natural killer cells, which can be used to reliablyidentify this subpopulation of peripheral blood mononuclear cells.

It is another object of the invention to provide antibodies that willbind to unique epitopes present on a cell surface structure selectivelyexpressed on functionally differentiated NK cells.

SUMMARY OF THE INVENTION

These as well as other objects and advantages are achieved in accordancewith the present invention, which provides a partially purifiedpreparation of a novel natural killer cell-specific molecule, tomonoclonal antibodies and immunoreactive fragments and derivativesthereof that bind to unique epitopes present on this NK cell-specificmolecule, and to hybridomas that produce the monoclonal antibodies.Methods of using the antibodies and fragments and derivatives are alsoprovided.

The novel NK cell-specific molecule of the invention consistsessentially of a pair of polydispersed glycoproteins, designated hereinas PEN5α ( and PEN5β, having apparent molecular weights of 120-150 and210-245 kdal, respectively, as determined by SDS polyacrylamide gelelectrophoresis on a 6% polyacrylamide gel under non-reducingconditions. The unique epitopes of the PEN5α/PEN5β glycoprotein pair arepreferentially expressed on the subpopulation of peripheral blood NKcells which are of the phenotype CD16 ⁺ CD56 ^(dim) relative to theirexpression on peripheral blood NK cells having the phenotype CD16 ⁺ CD56^(bright) and are not present on CD3 ⁺ T lymphocytes or CD20 ⁺ Blymphocytes. In preferred embodiments of the invention, the antibody isunreactive with peripheral blood T cells, activated T cells, thymocytes,peripheral blood B cells, splenic B cells, activated B cells, monocytes,granulocytes, platelets, and red blood cells. The antibodies of theinvention are preferably monoclonal antibodies and in particularlypreferred embodiments, are of mouse or human origin, or they arechimeric antibodies having at least the constant region thereof of humanorigin.

All monoclonal antibodies having the above specificity andcharacteristics are encompassed by the present invention. The monoclonalantibodies are produced by hybrid cell lines using conventionalhybridization and screening techniques, such as those described inAnderson et al, J. Immunol., 143:1899 (1989), which is herebyincorporated by reference. As is well known in the monoclonal antibodyart, independently produced hybrid cell lines that produce monoclonalantibodies specific for a given antigenic determinant are typicallydistinct from one another, as is each of the monoclonal antibodies soproduced. Thus, while repetition of the procedure described herein canresult in the production of a hybrid cell line that produces a usefulmonoclonal antibody in accordance with the invention, it is unlikelythat it will produce a hybrid cell line that produces a monoclonalantibody that is chemically an exact copy of the monoclonal antibodydescribed below.

In another embodiment of the invention, the epitope recognized by theantibodies of the invention is a sulfated polylactosamine carbohydraterelated to keratan sulfate glycosaminoglycan.

In yet another embodiment, the antibodies have the characteristics ofthe monoclonal antibody, alternatively referred to herein as eitheranti-PEN5 or mAb 5H10, secreted by a hybridoma identified by ATCCAccession No. HB11441.

The antibodies and/or immunoreactive fragments or derivatives of theinvention can be labeled, e.g. with a radioactive, enzymatic, orfluorescent label and used to detect, enumerate, and/or purifyfunctionally differentiated NK cells in a mixed population of cells andto distinguish these cells from non-NK cells and NK cells that are notfunctionally differentiated. Identification of the functionallydifferentiated subpopulation of NK cells involves (a) contacting asuitable sample that contains a mixed population of cells, which can be,for example, peripheral blood, bone marrow aspirate, or lymphoid tissue,with an antibody of the invention or an immimoreactive fragment orderivative thereof, and (b) detecting immune complex formation. Immunecomplex formation can be detected by any of the techniques that areconventional and well known in the art.

The antibodies of the invention can also be used to selectivelyeliminate functionally differentiated NK cells that are of the phenotypeCD16 ⁺ CD56 ^(dim) in a sample comprising a mixed population of cells.Thus, in another aspect of the invention, methods are provided forselectively eliminating or removing functionally differentiated naturalkiller cells from a suitable sample, preferably a biological sample,which involve (a) contacting the sample with an antibody of theinvention or an immunoreactive fragment or derivative thereof, which isoptionally linked to a radionucleotide or a toxin, and (b) removing fromthe sample the cells that bind to the antibody, fragment or derivative.In preferred embodiment of the invention, the biological sample is bonemarrow aspirate.

These as well as other features and advantages of the present inventionwill be apparent to persons skilled in the art from the followingdetailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1D comprise four two-color flow cytometry histograms,which collectively show that the PEN5 epitope is expressed selectivelyon CD56 ⁺ CD16 ⁺ peripheral blood lymphocytes (“PBL”). PBL were stainedby 2-color flow cytometry using rhodamine-conjugated anti-CD56 mAb,rhodamine-conjugated anti-CD3 mAb, rhodamine-conjugated anti-CD20 mAb,FITC-conjugated anti-CD16 mAb or biotinylated anti-PEN5 mAb. The bindingof biotinylated anti-PEN5 mAb was revealed using APC-conjugated avidin.Numbers in each quadrants indicate the percent of positive stainedcells.

FIGS. 2A through 2I comprise three sets of histograms that collectivelyshow the expression of the PEN5 epitope on distinct NK cell subsets. Forthe experiments that generated these histograms, purified NK cells wereeither unsorted, or sorted into CD56 ^(dim) and CD56 ^(bright) NK cellsubsets using rhodamine-conjugated anti-CD56 mAb and flow cytometry.Unsorted NK cells, CD56 ^(dim) and CD56 ^(bright) NK cells were furtheranalyzed for the expression of the PEN5 epitope using biotinylatedanti-PEN5 mAb and FITC-conjugated avidin. Controls were performed usingmouse isotype matched control mAb. The numbers in each histogramsindicate the percent of positive stained cells.

FIG. 3 comprises a series of flow cytometry histograms illustrating thekinetics of PEN5 expression on activated NK cells. Sorted CD56 ^(dim)and CD56 ^(bright) NK cells were activated for 20 days with ionomycinand lymphocyte conditioned medium as described in the examples. At theindicated period of time, (i.e, 0, 6, 8, 10, 14, and 20 days of culture)aliquots of the activated NK cell populations were analyzed for theircell surface phenotype by flow cytometry using isotype matched controlmAb, anti-CD56 and anti-PEN5 mAb. Results indicate the percent ofpositively stained cells (%); the total mean fluorescence intensity isindicated below.

FIG. 4 is a series of flow cytometry histograms that illustrate the cellsurface expression of the PEN5 epitope on leukemic NK cells. Peripheralblood NK cells, as well as peripheral blood mononuclear cells (PBMC)isolated from three patients undergoing granular lymphocyteproliferative disorder “GLPD” blast crisis (GLPD1-3) were analyzed forthe cell surface expression of CD56 and PEN5 using indirectimmunofluorescence and flow cytometry. The numbers in each histogramindicate the percent of positive stained cells.

FIG. 5 is a reproduction of an SDS gel from an immunoprecipitation ofPEN5 glycoproteins. Detergent lysates prepared from radioiodinated NKcells were immunoprecipitated using 5H10 mAb or mouse IgM control.Samples were then separated under non-reducing conditions on a 6%SDS-polyacrylamide gel.

FIG. 6 is a reproduction of an SDS gel from immunoprecipitationperformed after enzymatic deglycosylation of PEN5 glycoproteins.Detergent lysates prepared from radioiodinated NK cells wereimmunoprecipitated using 5H10 mAb. Affinity-purified PEN5α and PEN5βglycoproteins were eluted from the antibody-coated sepharose beads using0.15M NH₄OH, pH 10.5. Aliquots of this dried sample were then subjectedto deglycosylation for 24 hr at 37° C. using PNgase F (lane 6),O-glycanase (lane 3), keratanase I (lane 2), O-glycanase and keratanase(lane 4), neuraminidase (lane 6), and PNgase F and neuraminidase (lane7). Control eluates incubated in phosphate buffered saline (PBS) withoutany enzymes were separated in lane 1. Samples were separated undernon-reducing conditions on a 6-12% SDS-polyacrylamide gradient gel.

FIGS. 7A through 7C illustrate the reactivity of anti-PEN5 mAb withkeratan sulfate glycosaminoglycans.

In FIG. 7A, I¹²⁵-labeled 5H10 mAb (1×10⁶ cpm/sample) was preincubatedfor 20 min at 4° C. in PBS in the presence of the indicatedconcentrations of bovine cornea keratan sulfate (BC). The mixture wasthen added to NK cells for another 20 min incubation at 4° C., prior tothree washes in PBS-1% BSA. Samples were counted in a counter, andresults are expressed as mean cpm of duplicate samples (SD<10%). Whenused in incubation with NK cells or anti-PEN5 mAb, the followingcarbohydrates used at 10 mg/ml were without any effect on anti-PEN5binding to the NK cell surface: chondroitin sulfate B, heparin, heparansulfate, dextran sulfate, GlcNAc, mannose 6-phosphate, lactose,galactose-6-phosphate, fucose, glucose 6-phosphate, glucose andgalactose.

In FIG. 7B, peripheral blood NK cells were incubated in PBS-1% BSA for 3hr with glycosidases (0.025 U/ml) or 45 proteases (5 mg/ml) at 37° C.,respectively. Cell surface expression of the PEN5 epitope was thenanalyzed by flow cytometry using anti-PEN5 mAb. Percent modulation wascalculated as the ratio of the total linear mean fluorescence intensityof the treated cells over that of untreated control cells.

In FIGS. 7C through 7E, the antigenicity of anti-PEN5 mAb for aggrecanproteoglycans was analyzed by ELISA as described in the Examples. Theanti-keratan sulfate mAb 5D4 was used as a positive control.Chondroitinase ABC was used at 0.04 U/ml, keratanase I was used at 0.05U/ml and keratanase II was used at 0.004 U/ml, for 1 hr at 37° C. InFIG. 7C, the cross-hatched bars represent reactivity with the anti PEN5antibody, 5H10, while the open bars represent reactivity with thecontrol antibody, 5D4. Abbreviations used are: CD1=embryonic chickcartilage aggrecan; BNC=bovine nasal cartilage aggrecan; RC=swarm ratchondrosarcoma aggrecan; and SHK =shark cranial cartilage aggrecan.

FIGS. 8A through 8C illustrate the results of immunogold staining of thePEN5 epitope on NK cells. Peripheral blood NK cells were stained withanti-PEN5 mAb followed by gold-labeled anti-mouse IgM antibodies, andglutaraldehyde-fixed cells were then analyzed by transmission electronmicroscopy. Magnification: x48500, 0.972 cm=200 nm. The threephotomicrographs 8A-8C represent different views of the same stainedcell.

FIGS. 9A through 9H illustrate a comparative histochemical staining ofnormal adult lymph node and tonsil. Magnification of PEN5 staining shownFIGS. 9D and 9H is 40×. Magnification in all other panels is 10×.Monoclonal antibodies used to stain tissue sections, and specificmethods are described in the Materials and Methods found at Example 6.

FIGS. 10A through 10F illustrate comparative histochemical staining ofnormal adult and fetal thymus. Magnification of adult thymus stainedwith control and PEN5 specific antibodies is 20×. Magnification of allother panels is 10×. Monoclonal antibodies used to stain tissuesections, and specific methods are described in the Materials andMethods found at Example 6.

FIGS. 11A through 11H illustrate comparative histochemical staining ofnormal adult and fetal liver. Magnification of adult liver stained withanti-PEN5 is 20× (FIG. 11C) and 40× (FIG. 11D). Magnification of fetalliver stained with PEN5 is 10× (FIG. 11G) and 60× (FIG. 11H).Magnification of all other panels is 10×. Monoclonal antibodies used tostain tissue sections, and specific methods are described in theMaterials and Methods found at Example 6.

FIGS. 12A through 12F illustrate the comparative histochemical stainingof normal adult lung and colon. Magnification of lung stained withanti-CD56 and anti-PEN5 is 20×. Magnification of all other sections is10×.

FIGS. 13A through 13D illustrate dual labeling of tissue infiltratinglymphocytes. Tissue sections from adult spleen (panels A and B) or adultappendix (panels C and D) were double labeled with fluorescein taggedanti-PEN5 (panels A and C) and rhodamine tagged anti-TIA-1 (panels B andD), prior to examination by fluorescent microscopy. Black arrowheadsshow the location of PEN5 ⁺ cells in panels A and C. White arrowheadsshow the location of both PEN5 ⁺ cells and TIA-1 ⁺ cells in panels B andD.

DETAILED DESCRIPTION OF THE INVENTION

Natural killer cells are CD3:TCR⁻, CD16 ⁺, CD56 ⁺ large granularlymphocytes. Two functionally distinct populations of peripheral bloodNK cells can be differentiated by their surface expression of an isoformof the neural cell adhesion molecule, NCAM (also known as CD56). CD56^(bright) NK cells have the attributes of an undifferentiated cell inthat they proliferate vigorously in response to exogenous cytokines, butlargely lack cytolytic activity. CD56 ^(dim) NK cells have theattributes of a more differentiated cell, in that they proliferatepoorly in response to exogenous cytokines, but are potent cytolyticeffector cells. Several monoclonal antibodies that recognize human CD56are available commercially, for example from Coulter Corp. (Hialeah,Fla.) and AMAC, Inc. (Westbrook, Me. ).

NK cells are capable of mediating two types of cytotoxic effectorfunction: natural cytotoxicity and antibody-dependent cellularcytotoxicity (“ADCC”). In this capacity, NK cells play an important rolein host defense against viral infection, and in immune surveillanceagainst the establishment of transformed cells. More recent resultsindicate that NK cells can effect a primitive form of allorecognitionwhich can contribute to graft rejection during allogeneictransplantation and also to graft-versus-host disease. For thesereasons, the reliable identification of NK cells within the mononuclearcell population is of great importance. The present invention relates tothe identification and molecular characterization of a novel sulfatedpolylactosamine epitope whose expression is largely restricted to thefunctionally differentiated population of LGLs previously characterizedas CD16 ⁺ CD56 ^(dim), cytolytic effectors and to antibodies that arecapable of recognizing the same. Because this epitope is not expressedon resting or activated T cells, resting or activated B cells,monocytes, granulocytes, platelets or red blood cells, antibodies thatbind to unique epitopes on the PEN5 glycoprotein pair can be used todirectly identify this important population of mononuclear cells.Furthermore, because the epitope is preferentially expressed on thefunctionally differentiated subpopulation of NK cells relative to CD56^(bright) NK cells that have the attributes of an undifferentiated cell,antibodies that bind to the epitope can be used to distinguish these twosubpopulations of NK cells from one another.

Thus, in one aspect of the invention there is provided an antibody orfragment or derivative thereof, that recognizes a unique epitope of thePEN5 glycoprotein pair. As used herein, the phrase “unique epitope”means any epitope on the PEN5α glycoprotein and/or the PEN5βglycoprotein, which like the novel sulfated polylactosamine epitopeidentified herein, is present on a high percentage, e.g., at least about70%, of the population of LGLs previously characterized as CD56 ^(dim) ,CD16 ⁺ natural killer cells and on a significantly lower percentage ofthe population of NK cells that are phenotypically CD16 ⁺ CD56^(bright), but not on CD3 ⁺ T cells, or CD20 ⁺ B cells. The “uniqueepitope” may be present on a glycosylated form of the PEN5 glycoproteinpair as it is ordinarily expressed on the cell surface of CD56 ^(dim) ,CD16 ⁺ natural killer cells as previously described, or, the “uniqueepitope” may be present on an unglycosylated or deglycosylated form ofthe PEN5 glycoprotein pair. As used herein, the term “unglycosylated”means a PEN5 molecule where both the PEN5α and the PEN5β glycoproteinsare free of any covalently attached carbohydrate moieties. As usedheroin, the term “deglycosylated” means a PEN5 molecule where either orboth. of the PEN5α glycoprotein or the PEN5β glycoprotein is partiallyglycosylated but does not contain the same full contingent ofcarbohydrate moieties as the PEN5α glycoprotein or the PEN5βglycoprotein as it is ordinarily expressed on the cell surface of CD56^(dim) , CD16 ⁺ natural killer cells.

The antibodies, fragments, and derivatives of the invention are usefulas research reagents, to unambiguously identify, quantify and/or purifynatural killer cells in a mixed population of cells and in isolatingthese natural killer cells therefrom. The antibodies, fragments andderivatives of the invention may also be useful therapeutically, eitheralone, in combination with complement, or conjugated to a radioactivematerial or a toxin to treat disorders of the immune system where NKcells are implicated as mediators of disease, especiallygraft-versus-host disease and solid organ and allogenic bone marrowtransplant rejection. Monoclonal antibodies, and chimeric and humanizedantibodies, are preferred for detection and therapy, respectively.Antibodies, fragments and derivatives thereof recognizing a uniqueepitope on a deglycosylated or unglycosylated form of the PEN5 molecule,may be useful in the diagnosis and treatment of immune disordersassociated with NK cells expressing PEN5 exhibiting an aberrantglycosylation pattern compared to PEN5 normally expressed on NK cells.

In the following description, reference will be made to variousmethodologies known to those of skill in the art of immunology, cellbiology, and molecular biology. Publications and other materials settingforth such known methodologies to which reference is made areincorporated herein by reference in their entireties as though set forthin full.

Preparation And Research Uses Of Antibodies

Monoclonal antibodies of the invention can be prepared using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the original techniques of Köhler and Milstein, Nature, 265:495-497(1975), modified as described in Anderson et al, J. Immunol., 143:1899(1989), the pertinent portions of which are hereby incorporated byreference and the more recent human B cell hybridoma technique andEBV-hybridoma technique well known to persons skilled in the art.

As part of the production of the monoclonal antibodies of the invention,various host animals, including but not, limited to rabbits, mice,hamsters, and rats can be immunized by injection with NK cells thatexpress the PEN5 glycoprotein and, after a sufficient time, the animalis sacrificed and spleen or other immune cells obtained. The preferredimmunogen to be used in the immunization protocol is a preparation offreshly isolated NK cells, purified from peripheral blood lymphocytes bynegative selection. Other immunogens that alternatively could be usedinclude partially purified preparations of the PEN5 molecule, the PEN5αglycoprotein or the PEN5β glycoprotein, including the glycosylated,deglycosylated or unglycosylated forms thereof and derivatives andfragments thereof. A partially purified preparation of the PEN5 moleculecan be prepared from permeabilized NK cells followingimmunoprecipitation and SDS gel electrophoresis using 6% polyacrylamidegel as hereinafter described using techniques well known to personsskilled in the art. However, any suitable method for partially purifyingthe PEN5 molecule or the PEN5α or the PEN5β glycoprotein as describedabove can be satisfactorily employed and alternative methods of partialpurification will be readily apparent to those persons skilled in thisarea of technology. Once the protein core(s) of the PEN5α and PEN5βmolecules have been cloned, recombinantly produced molecules can also beused as an immunogen. The spleen or other immune cells obtained from theanimal are immortalized by fusing the spleen cells with an immortalizedcell line, generally in the presence of a fusion enhancing reagent, forexample, polyethylene glycol. The resulting cells, which include thefused hybridomas, are then allowed to grow in a selective medium, suchas HAT medium, and the surviving cells are grown in such medium usinglimiting dilution conditions. The cells are grown in a suitablecontainer, e.g., microtiter wells, and the supernatant is screened formonoclonal antibodies having the desired specificity.

In a preferred embodiment, the monoclonal antibodies of the presentinvention are prepared as described in the Examples.

Screening procedures that can be used to screen hybridoma cellsproducing antibodies to a PEN5 epitope include, but are not limited to,(1) enzyme-linked immunoadsorbent assays (ELISA), (2)immunoprecipitation and (3) fluorescent activated cell sorting (FACS)analyses. Many different ELISAS that can be used to screen for anti-PEN5monoclonal antibodies can be envisioned by persons skilled in the art.These include but are not limited to formats comprising purified orrecombinantly produced PEN5 glycoproteins attached to a solid phase orformats comprising the use of freshly isolated whole NK cells or celllysate membrane preparations either attached to the solid phase or boundto antibodies attached to the solid phase. Samples of hybridomasupernatants would be reacted with either of these two formats, followedby incubation with, for instance, goat-anti-mouse immunoglobulincomplexed to an enzyme-substrate that can be visually identified.

Initial screening is preferably conducted by screening hybridomasupernatants by flow cytometry for their reactivity with NK cells, butnot with T cells, B cells, and monocytes. Further characterization ofthe hybridomas for those that produce monoclonal antibodies that arepreferentially expressed on NK cells that are phenotypically CD16 ⁺ CD56^(dim) relative to NK cells that are phenotypically CD16 ⁺ CD56^(bright) can be conducted by testing on purified populations oflymphoid and non-lymphoid cells by indirect immunofluorescence assaysand flow cytometry, substantially as described in the Examples herein.Monoclonal antibodies that recognize a PEN5 epitope that ispreferentially expressed on functionally differentiated NK cells willreact with an epitope that is present on a high percentage NK cells thatphenotypically are CD56 ^(dim) CD16 ⁺ cells, e.g., at least about70-90%, preferably about 80%, of such cells, and with a much lowerpercentage of NK cells that are phenotypically CD16 ⁺ CD56 ^(bright)(e.g., about 10 to 35%), but will not react with CD3 ⁺ T cells or CD20 ⁺B cells. In preferred embodiments, the antibody will also be unreactivewith monocytes, granulocytes, platelets, and red blood cells. Monoclonalantibodies that compete with the 5H10 antibody in competition assayswell known to persons skilled in the art are likely to recognizeessentially the same epitope as mAb 5H10, while monoclonal antibodiesthat fail to compete with mAb 5H10 but it nevertheless meet the criteriaof being unique to the CD16 ⁺, CD56 ^(dim) subpopulation of NK cells arelikely to recognize a different epitope on the PEN5 glycoprotein pair.Both classes of antibodies are considered within the scope of thepresent invention.

Once the desired hybridoma has been selected and cloned, the resultantantibody may be produced in one of two major ways. The purest monoclonalantibody is produced by in vitro culturing of the desired hybridoma in asuitable medium for a suitable length of time, followed by the recoveryof the desired antibody from the supernatant. The length of time andmedium are known or can readily be determined. This in vitro techniqueproduces essentially monospecific monoclonal antibody, essentially freefrom other species of anti-human immunoglobulin. However, the in vitromethod may not produce a sufficient quantity or concentration ofantibody for some purposes, since the quantity of antibody generated isonly about 50 μg/ml.

To produce a much larger quantity of monoclonal antibody, the desiredhybridoma may be injected into an animal, such as a mouse. Preferablythe mice are syngeneic or semi-syngeneic to the strain from which themonoclonal-antibody producing hybridomas were obtained. Injection of thehybridoma causes formation of antibody producing tumors after a suitableincubation time, which will result in a high concentration of thedesired antibody (about 5-20 mg/ml) in the ascites of the host animal.

Antibody molecules can be purified by known techniques, e.g. byimmunoabsorption or immunoaffinity chromatography, chromatographicmethods such as high performance liquid chromatography or a combinationthereof.

Following these protocols, any person skilled in this area of technologycan readily isolate hybridomas that produce monoclonal antibodiesexhibiting specificity for a unique epitope on functionallydifferentiated natural killer cells. Although only a single hybridomaproducing a monoclonal antibody (5H10) against the human PEN5 epitope isexemplified by way of working example, it is contemplated that thepresent invention encompasses all monoclonal antibodies exhibiting thecharacteristics of mAb 5H10 as herein described.

For example, it was determined that the subject monoclonal antibody 5H10belongs to the class IgM. However, a monoclonal antibody exhibiting thecharacteristic described herein may be of class IgG, subclass IgG₁,IgG₂α, IgG₂β, or IgG₃, or of classes IgM, IgA, or other known Igclasses. The differences among these classes or subclasses will notaffect the selectivity of the reaction pattern of the antibody, but mayaffect the further reaction of the antibody with other materials, suchas (for example) complement or anti-mouse antibodies. Although thesubject antibody is specifically IgM, it is contemplated that antibodieshaving the patterns of reactivity illustrated herein are included withinthe subject invention regardless of the immunoglobulin class or subclassto which they belong.

Moreover, while the specific example of the novel antibody of thepresent invention is from a zurine source, this is not meant to be alimitation. The above antibody and those antibodies having thecharacteristics of the mAb 5H10, whether from a mouse source, othermammalian source including human, rat, or other sources, or combinationsthereof, are included within the scope of this invention, as set forthabove.

The antibodies may be used for the detection and enumeration by indirectstaining of CD16 ⁺, CD56 ^(dim) subpopulation of NK cells in normalindividuals or in disease states, for example by fluorescencemicroscopy, flow cytometry, immunoperoxidase, or other indirectmethodologies. Panning techniques are also possible. The antibodies mayalso be used for purification of human natural killer cells which areCD16 ⁺, CD56 ^(dim).

Preparation of Fragments and Derivatives of Antibodies

Also included within the scope of the present invention are antibodyfragments and derivatives which comprise at least the functional portionof the antigen binding domain of an anti-PEN5 antibody molecule.

Antibody fragments which contain the binding domain of the molecule canbe generated by known techniques. For example, such fragments include,but are not limited to: the F(ab′)₂ fragment which can be produced bypepsin digestion of the antibody molecule; the Fab′ fragments which canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragment,and the Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent. See, e.g., NationalInstitutes of Health, 1 Current Protocols In Immunology, Coligan et al.,ed. § 2.8, 2.10 (Wiley Interscience, 1991).

Antibody fragments also include Fv fragments, i.e., antibody products inwhich there are no constant region amino acid residues. Such fragmentscan be produced, for example as described in WO 92/04381 or U.S. Pat.No. 4,642,334.

When antibodies produced in non-human subjects are used therapeuticallyin humans, they are recognized to varying degrees as foreign and animmune response may be generated in the patient. One approach forminimizing or eliminating this problem, which is preferable to generalimmunosuppression, is to produce chimeric antibody derivatives, i.e.antibody molecules that combine a non-human animal variable region and ahuman constant region. Chimeric antibody molecules can include, forexample, the antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimeric antibodies have been described and can be used to makechimeric antibodies containing the immunoglobulin variable region whichrecognize a unique epitope on the PEN5 antigen. See, for example,Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851 (1985); Takeda etal., Nature 314:452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567;Boss et al. U.S. Pat. No. 4,816,397; Tanaguchi et al., Bur. Patent Pub.EP171496; Eur. Patent Pub. 0173494; United Kingdom Patent GB 2177096B.Such chimeras produce a less marked immune response than non-chimericantibodies.

For human therapeutic purposes, the monoclonal or chimeric antibodies ofthe invention can be further humanized by producing human constantregion chimeras, in which even parts of the variable regions, especiallythe conserved or framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such altered immunoglobulin molecules may be made by any of severaltechniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci.U.S.A., 80:7308-7312 (1983); Kozbor et al., Immunology Today, 4:7279(1983); Olsson et al., Meth. Enzymol., 92:3-16 (1982)), and arepreferably made according to the teachings of PCT Pub. WO 92/06193 or EP0239400. There are also a number of companies that humanize antibodiescommercially, for example Scotgen Limited, 2 Holly Road, TwickenbAm,Middlesex, Great Britain.

These humanized antibodies are preferable for immunotherapy in that theyminimize the effects of an immune response. This in turn leads to alowering of any concomitant immunosuppression and to include increasedlong term effectiveness in, for instance, chronic disease situations orsituations requiring repeated antibody treatments.

Antibody Conjugates For Detection and Therapy

In addition to molecular antibody fragments and derivatives, antibodyderivatives or immunoconjugates consisting of an antibody molecule orbinding region thereof bound to a label such as a radioisotope,fluorescent tag (e.g., fluorescein isothiocyanate, phycoerythrin,phycoerythrin Cy5, or rhodamine), enzyme (e.g., biotin), or other tracermolecule can be made by techniques known in the art. Alternatively, theantibody molecule or fragment thereof can be bound to a therapeuticallyuseful biological or chemical molecule targeted to its desired site ofaction by virtue of the antibody's binding specificity. As one exampleof such an embodiment, a cytotoxic compound can be conjugated to anantibody of the invention which is specific for NK cells which are thecausative agents of an immune disorder, for example, bone marrow graftrejection. The cytotoxic compound, which can be for example, aradionucleotide or a toxin, such as diphtheria toxin, in conjugated formis thus targeted to the implicated NK cells.

Immuoassays

The antibodies of the invention and the fragments and derivativesthereof containing the binding region (e.g., Fab, Fab′, F(ab′)₂), can beused in various immunoassays. Such immunoassays include, but are notlimited to, competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, and immunoelectrophoresis assays,to name but a few.

Differentiated NK cells, especially human NK cells, can be detected in abiological sample including a mixed population of cells, for example,hematopoietic and lymphoid cells, using the antibodies, fragments orderivatives. Suitable biological samples include peripheral blood, bonemarrow aspirate and lymphoid tissue. When used in an assay as described,the antibody is typically labeled so that its binding with the relevantNK cell subpopulation can be detected. Any suitable label well known topersons skilled in the art, including but not limited to fluorescentdyes, radioactive isotopes, enzymes which catalyze a reaction producingdetectable products, biotin, or metal ions detectable by nuclearmagnetic resonance can be employed.

Therapeutic Applications of NK Cell-Specific Antibodies, Fragments andDerivatives

Bone marrow transplantation is increasingly used for the treatment ofdisorders of the immune system, aplastic anemia, and especiallyhematopoietic malignancies, such as acute lymphocytic leukemia. For manyyears, graft-versus-host disease (“GVHD”) and its attendantcomplications have presented a serious problem in human bone marrowtransplantation. When it became apparent that T cells contained withinthe bone marrow inoculum are effectors of GVHD, many bone marrowtransplant programs resorted to using T-cell depleted bone marrow cells.This procedure has somewhat successfully reduced the incidence andseverity of GVHD. However, several new problems have emerged as a resultof T cell depletion, lid including an increased incidence of bone marrowgraft rejection. Furthermore, GVHD continues to be a serious problem inmany bone marrow transplantation recipients, especially in non-T celldepleted transplants.

Several lines of evidence have directly implicated natural killer cellsin graft rejection, and more recently, in graft-versus-host disease. Forexample, treatment of recipients with an antiserum specific for NK cells(Lotzova et al, Transplantation, 35:490 (1983)) ablated allograftresistance and injection of recipients with NK clones caused allograftrejection in NK cell deficient beige mice which do not manifest marrowgraft rejection. Warner et al., Nature 300:31 (1982). See also, Martinet al, Advances Immunol., 40:379-431 (1987), Yu et al Amer. Rev.Immunol., 10:189-213 (1992) and Moretta et al, Immunol. Today,13:300-305 (1992).

The scientific literature also suggests that NK cells may play adeleterious role in graft-versus-host disease (“GVHD”) following solidorgan or tissue transplants. (Ferrara et al, Transplantation, 47:50-54(January, 1989); MacDonald and Gartner, Transplantation, 54:147-151(July, 1992)). See also, U.S. Pat. No. 4,772,552.

Since the antibodies of the invention can be used to target thefunctionally differentiated subpopulation of NK cells specifically, theinvention may also be useful prophylactically and therapeutically, inthe prevention and treatment of graft rejection in solid organ and bonemarrow transplantation, and in graft-versus-host disease, by modulatingthe function and number of cytolytic effector NK cells in vivo. Althoughit is contemplated that the anti-NK cell-specific antibodies andfragments and derivatives thereof will have applicability for animalsubjects in addition to human beings, such as domesticated animals, thetherapeutic aspects of the invention are of the greatest value in thetreatment of disorders in humans.

For example, in bone marrow transplantation, the antibodies, fragmentsand derivatives of the invention can be used to remove the CD16 ⁺ CD56^(dim) cytolytic effector population of cells from bone marrow aspiratesex vivo, prior to transplantation of the marrow into the marrowrecipient. Removal of these natural killer cells from the bone marrowaspirate can be accomplished by conventional methods, such as those usedin immunological T cell depletion. Antibodies that exhibit the abilityto lyse NK cells in the presence of complement can be used incombination with complement to treat the bone marrow ex vivo prior totransplantation, to kill the NK cells that might otherwise contribute tothe etiology of graft-versus-host disease in the recipient.Alternatively, the anti-PEN5 antibody might be linked to a toxin asdescribed, to kill the cytolytic effector NK cells. The antibodies,fragments or derivatives of the invention could also be administered tothe bone marrow recipient in vivo prior to the transplantationprocedure.

The selective in vivo removal of NK cells may also prove useful in thetreatment of autoimmune diseases such as SLE, which are in part mediatedby NK cells.

The antibodies, fragments, or derivatives of the invention may also beuseful in the prophylaxis and/or treatment of solid organ graftrejection and bone marrow rejection, especially in allogeneic bonemarrow transplant recipients where T cell depletion has been employed.

When used prophylactically or therapeutically in vivo, the NK-specificantibodies, fragments or derivatives of the invention may be useful inunmodified form for modulating the number and function of the cytolyticeffector population of NK cells, or they can be conjugated toradionucleotides or toxins by means well known in the art and used todeliver the conjugated substance to deleterious NK cells for negativemodulation. Non-limiting examples of radionucleotides which can beconjugated to antibodies and administered include ²¹²Bi, ¹³¹I, ¹⁸⁶Re,and ⁹⁰Y. These elements exert their effect by locally irradiating thecells, leading to various intracellular lesions, well known to personsskilled in the art of radiotherapy.

Cytotoxic drugs that can be conjugated to antibodies and administeredfor in vivo therapy include, but are not limited to, daunorubicin,doxorubicin, methotrexate, and mytomycin C. For a more detaileddiscussion of these classes of drugs and their mechanisms of action,see, Goodman et al., Goodman and Gilman's The Pharmaceutical Basis OfTherapeutics, 8th ed. Pergamnn Press (1991).

As an example of conjugation to a toxin, an anti-PEN5 monoclonalantibody can be combined with diphtheria toxin, by the method of Bumol,Proc. Natl. Acad. Sci., 80:529 (1983). Briefly, monoclonal antibodiesreactive with an NK cell specific epitope are prepared as described byBumol. The antibodies are purified and combined with excess (6 mol/mol)N-succinimydyl 3-(2-pyridyldithio) propionate (Pharmacia, Uppsala,Sweden) in PBS. After 30 minutes incubation at room temperature, thesolution is dialyzed against PBS. The modified antibodies are conjugatedwith an appropriate toxin, such as diphtheria toxin A chain. Othertoxins such as ricin A can also be employed. The diphtheria toxin Achain is isolated as detailed in Bumol, supra. The modified antibodiesare mixed with excess (3 mol/mol) reduced diphtheria toxin A chain (10%of the total volume), allowed to react for 36 hours at 4° C., andconcentrated by chromatography on Sephadex G-2000. The product isapplied to a Sephadex G200 column (1.0×100 cm), allowed to equilibrateand eluted with PBS.

Using these and other similar techniques known to persons in the art,the effector population of natural killer cells can be selectivelyeliminated in the transplant recipient.

The route of administration for the in vivo therapeutic modalities mayinclude intrade-mal, intramuscular, intraperitoneal, intravenous, orsubcutaneous injection, intranasal routes and slow release forms, suchas those delivered in transplantable forms, on patches or in othercolloidal forms. In one embodiment, the antibody can be encapsulated inliposomes.

The effective dose of the therapeutic reagent will be a function of theparticular reagent employed, the presence and nature of conjugatedtherapeutic reagent, the patient, and his or her clinical condition.Effective doses of the antibodies, fragments, or derivatives of theinvention for use in preventing, suppressing, or treating animmune-related disease are in the range of about 1 ng to 100 mg/kg bodyweight. A preferred dosage range is between about 10 ng and 10 mg/kg,and a more preferred dosage range is between 100 ng and 1 mg/kg.

Various pharmacologic compositions may be utilized in order to deliverthe antibodies, or fragments or derivatives thereof, according to theinvention. Any suitable pharmaceutical agent with desirable solubilitycharacteristics and chemical properties may be used, including but notlimited to, where appropriate, saline or dextrose solutions. The reagentitself must be properly formulated, for example, as a humanized orchimeric antibody combined with various buffers, sugars, or stabilizingcompounds that increase the stability or half life of the antibody. Toextend the half-life, the reagent can first be modified to increase ordecrease the amount of carbohydrate complexed to it, or alternatively,can be complexed with a reagent such as polyethylene glycol. Finally,pharmaceutical compositions comprising the therapeutic reagent in theappropriate buffers, salts, and pH are required.

Therapeutic kits can comprise the therapeutic compositions of theinvention in one or more containers.

The PEN5α/PEN5β Glycoprotein Pair

The invention also provides partially purified preparations of the NKcell-specific molecule, called PEN5α/PEN5β, that is preferentiallyexpressed on the subpopulation of NK cells previously characterized asCD16 ⁺ CD56 ^(dim) NK cells. The molecule consists essentially of twomembrane bound glycoproteins.

As used herein, use of the term “partially purified preparation” withrespect to the PEN5 means the PEN5 molecule, consisting essentially ofthe PEN5α and PEN5β glycoprotein pair as herein described, which hasbeen purified from permeabilized NK cells following immunoprecipitationand SDS gel electrophoresis using 6% polyacrylamide gel as hereinafterdescribed. After the glycoproteins are fractionated on a gel, they canbe recovered and renatured in accordance with known and establishedtechniques.

As expected of a marker of functional differentiation, expression of thePEN5 epitope is down-modulated by stimuli which induce NK cellproliferation, and is largely absent from the leukemic NK cells ofpatients with granular lymphocyte proliferative disorder.

Immunoprecipitations of freshly isolated human NK cell detergent lysateswith mAb 5H10 revealed that the molecule consists essentially of twodistinct glycoproteins and also revealed that the average molecularweight of the larger species, PEN5α, is 227 ±4 kDa (n=12). The molecularweight range of the polydispersed PEN5α species was 210±3 kDa to 245±5kDa. The average molecular weight of the smaller species, PEN5β, was140±3 kDa, with a range of 123±3 kDa to 170±4 kDa. The migration of bothPEN5α and PEN5β as polydispersed bands suggests that both species arehighly glycosylated.

Enzymatic deglycosylation indicates that both PEN5α and PEN5β are 80-90%carbohydrate by weight. This result raised the possibility that theseproteins are either proteoglycans or mucin-type glycoproteins.

Proteoglycans are high molecular weight glycoproteins in which specificglycosaminoglycans are bound to proteins via Gal-xylose-Ser linkages[Bhavanandan, Glycobiology, (1991)], or in the case of keratan sulfatechains, terminal galactosamine linkages to serine or threonine. Thestudies described in the Examples below reveal that PEN5 molecules arefree of xylose-linked carbohydrates. However, the anti-PEN5 mAbs arereactive with sulfated polylactosamine carbohydrates present on keratansulfate glycosaminoglycans, which raised the possibility that PEN5αand/or PEN5β glycoproteins may be cell surface-associated keratansulfate proteoglycans.

Two types of keratan sulfate proteoglycans have been described:cartilage-type keratan sulfate proteoglycans are O-linked glycoproteins,whereas cornea-type keratan sulfate proteoglycans are N-linkedglycoproteins. Since PEN5α is an N-linked glycoprotein, it is possiblethat PEN5α is an unusual cell surface cornea-type keratan sulfateproteoglycan. Similarly, PEN5β is an O-linked glycoprotein sensitive tokeratanase treatment, and may be a cartilage-type keratan sulfateproteoglycan. However, the inability of six distinct anti-keratansulfate mabs to bind to NK cells, coupled with the lack of detection ofS³⁵sulfur-labeled material in 5H10 (anti-PEN5) immunoprecipitatesprepared from S³⁵ sulfur metabolically-labeled NK cells, indicate thatthe PEN5 glycoproteins are not keratan sulfate proteoglycans.

Alternatively, it has been reported that mucin-type glycoproteinssecreted by cultured hamster tracheal epithelial cells are sensitive tokeratanase I treatment and contain polylactosamine carbohydrates [Wu,Biochem J., 277:713 (1991)]. Mucin-type glycoproteins are highlyglycosylated proteins containing a majority of O-linkedoligosaccharides, and are associated with the cell membrane in a numberof cell types [carraway, Glycobiology 1:131 (1991); Strous, Rev. BiochemMol. Bio. 27:57 (1992); Devine, 35 (1992). Classification of PEN5β as anNK cell specific membrane-bound mucin-type glycoprotein is mostconsistent with our data. By contrast, the high content of N-linkedcarbohydrates in PEN5α is not consistent with its classification as amucin-type glycoprotein. Therefore the PEN5α:PEN5β complex appears to beanalogous to the ASGP-1:ASGP-2 complex derived from ascitic mammaryadenocarcinoma cells [Sherblom, J. Biol. Chem., 225:12051 (1980) inwhich only one component (ASGP-1) of the complex is a mucin-typeglycoprotein. The developmentally regulated PEN5β mucin-likeglycoprotein, like other cell surface mucins, may contribute tocytoprotection during lymphocyte mediated cytolysis.

The biochemical features of PEN5 molecules points the way for futureresearch. First, carbohydrates are major mediators of cell-cellinteractions [Jessel, Annu. Rev. NeuroSci., 13:227 (1990)]. Inparticular, ligands for E- and P-selectins have been shown to containeither sialyl-CD15 or CD57 polylactosamine epitopes [Philips, Science,250:1130 (1990); Larsen, Cell, 63:467 (1990); Needham, PNAS, 90:927(1993)], and GlyCAM-1 a membrane bound mucin glycoprotein is the ligandfor L-selectin [Lasky, 1992 #1853]. The NK cell surface expression ofthe sulfated polylactosamine PEN5 epitope, as well the mucin-likebiochemical characteristics of PEN5β, raise the possibility that PEN5glycoproteins contribute to NK cell specific adhesion. Second, in theirprotease-resistance as well as their extended rod-like structure, thePEN5 glycoproteins resemble epithelial cell mucins. The mucin-typeglycoproteins serve a protective role on the epithelial cell surface,and have been shown to protect cells from attack by cytotoxiclymphocytes. The PEN5 glycoproteins may therefore protect NK cells fromtheir own cytolytic machinery. The selective expression of PEN5 proteinson the terminally differentiated subset of NK cells would be consistentwith their acquisition of fully competent cytotoxic function. Exogenousmucins have been shown to inhibit NK cell killing, supporting theirpotential involvement in resistance to NK cell cytolytic functions[Ogata, Cancer Res., 52:4741 (1992)].

The PEN5 antigen can be used in preparing and/or purifying theantibodies of the invention and should also be useful in identifying thenatural counter-receptor for the PEN5 antigen on target cells. Aminoacid sequence information obtained from the PEN5 glycoprotein pair canalso be used to clone the PEN5α and PEN5β glycoprotein chains inaccordance with established techniques.

Deposit Information

Samples of the hybridoma (designated herein as 5H10) that secretesanti-NK cell-specific mouse monoclonal mAb 5H10 were deposited with theAmerican Type Culture Collection, (“ATCC”) 10801 University Blvd.,Manassas, Va. 20110 on Aug. 19, 1993 under the terms of the BudapestTreaty and assigned ATCC accession number HB11441. Without admittingthat access to the hybrid cell line is necessary to practice the claimedinvention. Applicants represent that the ATCC is a depository affordingpermanence of the deposit and ready accessibility thereto by the publicif a patent is granted. All restrictions on the availability to thepublic of the material so deposited will be irrevocably removed upon thegranting of a patent. The material will be available during the pendencyof the patent application to one determined by the Commissioner to beentitled thereto under 37 CFR 1.14 and 35 USC 122. The depositedmaterial will be maintained with all the care necessary to keep itviable and uncontaminated for a period of at least five years after themost recent request for the furnishing of a sample of the depositedmicroorganism, and in any case, for a period of at least thirty (30)years after the date of deposit or for the enforceable life of thepatent, whichever period is longer. Applicants and their assigneeacknowledges their duty to replace the deposit should the depository beunable to furnish a sample when requested due to the condition of thedeposit.

The invention will be more fully understood from the following

EXAMPLES

Abbreviations

The following abbreviations are used throughout the Examples reproducedbelow: BCK: bovine cornea keratan sulfate; BNC: bovine nasal cartilageaggrecan; CD1: embryonic chick cartilage aggrecan; LCM:leucocyte-conditioned medium; GLPD: granular lymphocyte proliferativedisorder; RC: Swarm rat chondrosarcoma aggrecan; SHK: shark cranialcartilage aggrecan.

Materials and Methods

The following methods and materials apply to Examples 1-5.

Reagents

Peptide-N-glycosidase (PNgase F) and Endo-a-N-acetylgalactosaminidase(O-glycanase) were used in the buffer provided by the manufacturer(Oxford Glycosystems). Keratanase I (keratan sulfate 1,4b-D-galactanohydrolase; ICN Biomedicals, (Costa Mesa, Calif.),keratanase II which attacks oversulfated forms of (keratan sulfate 1,4b-D-galactanohydrolase; ICN Biomedicals, (Costa Mesa, Calif.),keratanase II which attacks oversulfated forms of keratan sulfateresistant to keratanase I (Seikagaku America, Rockville, Md.) andneuraminidase (Calbiochem) were used in either PBS, PNgase F orO-glycanase buffers. Chondroitinase ABC (ICN Biomedicals,) was used insodium acetate 0.05M pH 7.4. Bovine cornea keratan sulfate (BC), as wellas other glycosaminoglycans and carbohydrates were purchased from Sigma,(St. Louis, Mo.)., Trypsin, chymotrypsin and pronase E were alsoobtained from Sigma. FITC- and PE-conjugated avidin were obtained fromBecton-Dickinson, (Paramus, N.J.).

Antibodies

Mouse monoclonal antibodies (mAb) reactive with CD2 (T11.1, IgG1), CD3(RW24B6, IgG2b), CD56 (N901, IgG1), CD20 (B1, IgG1) were obtained fromCoulter Corp., as well as isotype matched control mouse mAb (IgG andIgM). Radioiodination of PEN5 mAb was performed using lodobeads (Pierce)as previously described [Vivier, J. Immunol. 132:1410 (1991)]. Thecharacterization of the anti-CD16 mAb (3G8, IgG1), and the anti-keratansulfate mAb 5D4 (IgM) was reported elsewhere [Perussia, J. Immunol.,134:1410 (1984), Caterson, J. Biol. Chem., 258:8848 (1983)]. Thefollowing mAb recognize distinct epitopes on most keratan sulfatechains: 1B4 (IgG), 2D3 (IgG), 3D2 (IgM), 4D1 (IgM) and 8C2 [Sorrell, J.Invest. Dermatol. 95:347 (1990)]. FITC-labeled goat anti-mouse Ig(G+M)was purchased from Tago.

Cells

All cells were cultured in final medium consisting of RPMI 1640(Whittaker Bioproducts, (Walkersville, Md.) supplemented with 10% fetalcalf serum, 1 mM sodium pyruvate, 2 mM glutamine and 50 mg/mlgentamicin, all obtained from Gibco, (Grand Island, N.Y.) Purified NKcells and T cells were isolated from peripheral blood mononuclear cells(PBMC) obtained from healthy volunteers by negative selection usingimmuno-magnetic bead depletion [Vivier, Int. Immunol., 4:1313-1323(1992)]. In some experiments, NK cells and NK cell subsets (CD56^(bright) and CD56 ^(dim)) were further purified by flow cytometricsorting on an Epics V flow cytometer (Coulter Electronics) afterstaining with anti-CD56 mAb. Activation of NK cells was performed usingionomycin (1 μM) and 20% lymphocyte-conditioned medium (LCM) asdescribed previously [Robertson, J. Immunol., 150:1705 (1993)]. PBMCfrom three patients with a CD3:TCR⁻, CD16 ⁺, CD56 ⁺ granular lymphocyteproliferative disorder (GLPD) [Oshimi, Leukemia, 2:617 (1988)] wereisolated by Ficoll-Hypaque gradient centrifugation.

Immunoprecipitations

Cells were resuspended in PBS and subjected to radioiodination using125I by the lactoperoxidase method [Vivier, 1991 #1031]. After threewashes in PBS, cells were solubilized in NP-40 lysis buffer (1% NP-40,150 mM NaCl, 50 mM Tris HCl, pH 8.0, 1 mM PMSF, 10 μg/ml leupeptin, 10μg/ml aprotinin, 10 μg/ml AEBSF) for 15 min on ice. After removinginsoluble material by centrifugation at 12,000 rpm for 15 min,radioiodinated lysates were diluted in 1 ml lysis buffer and precleanedthree times with 3 μl of affinity-purified rabbit anti-mouse IgM or IgG(RAM, Jackson Immunoresearch Laboratories, (West Grove, Pa.) and 50 mlof a 50% solution of protein A-sepharose beads (Pharmacia, Milwaukee,Wis.). The immunoprecipitations were performed using 3 ml of theindicated mAb, 3 μl of RAM and 50 μl of protein A-Sepharose beads at50%. Sepharose-bound immune complexes were washed four times in lysisbuffer, and eluted either directly into sample buffer (2% SDS, 10%glycerol, 0.1M Tris-HCl, pH 6.8, 0.02% bromophenol blue) prior toelectrophoretic separation, or in elution buffer (0.15M NH₄OH, pH 10.5)prior to deglycosylation experiments.

Deglycosylation of radioiodinated PEN5

Radioiodinated PEN5 samples eluted from 5H10-coated Sepharose beads,were dried under vacuum and resuspended in appropriate deglycosylationenzyme buffers. The following enzymes were used alone or in combination:PNgase F (310 U/mi), O-glycanase (0.06 U/mi), keratanase I (0.25 U/ml)and neuraminidase (0.2 U/ml).

ELISA for aggrecan-type proteoglycans

Wells of microtiter plates were incubated with 10 μg/ml solutions of theindicated aggrecan-type proteoglycans overnight at 4° C. After washing,wells were incubated with 0.1M Tris, pH 7.6 containing 1% BSA or withthe indicated enzymes in this buffer. Following enzymatic digestion, astandard ELISA was performed using 1/1000 dilution of 5H10 (anti-PEN5)and 5D4 (anti-keratan sulfate), and 1/500 dilution of anti-mouse Ig(G+M)conjugated with alkaline phosphatase. Color was developed usingp-nitrophenyl phosphate substrate in 0.86M diethanolamine, pH 9.8. Allabsorbance values are the mean of 4 wells (SD<10%) and have beencorrected for non-specific binding of the second antibody.

Transmission electron microscopy

Peripheral blood NK cells were first stained using 5H10 (anti-PEN5) andcolloidal gold-labeled goat anti-mouse IgM (Amersham). After fixationusing % glutaraldehyde, the stained cells were examined by transmissionelectron microscopy.

EXAMPLE 1

Preparation And Characterization Of anti-PEN5 Antibody

In order to identify novel cell surface structures selectively expressedon NK cells, we generated a panel of mouse mAb (anti-PEN mAb) thatrecognized NK cells but not T cells. These antibodies were produced byimmunizing BALB/c mice with digitonin permeabilized peripheral blood NRcells as previously described [Anderson, J. Immunol. 143:1889 (1989)].Briefly, mononuclear cells were isolated from leukopheresis residues(obtained from normal blood donors at the Dana-Farber Cancer InstituteBlood Bank) by centrifugation over ficoll. These cells were cultured inplastic flasks in RPMI media containing 10% fetal calf serum for six totwelve hours to allow the adherence of monocytes. Nonadherent cells wereincubated with monoclonal antibodies reactive with CD5 (24T6G12, IgG2A),CD3 (RW24B6, IgG1), CD20 (B1H299, IGG2A), CD24 (MY4322A-1, IgG2B) atoptimal concentrations for thirty minutes, then washed extensively.Following the addition of magnetic beads coupled to goat and anti-mouseIg (Advanced Magnetics, Inc., Cambridge, Mass.) these populations weredepleted of T cells, B cells, monocytes by negative selection using amagnet. The remaining cells which were enriched for NK cells werephenotypically less than 5% CD3 ⁺, 75-95% CD56 ⁺, and 65-80% CD16 ³⁰ asdetermined by flow cytometry using an Epics profile (CoulterElectronics, Hialeah, Fla.). These cells were then permeabilized withdigitonin as described in Anderson, J. Immunol. 143:1889 (1989).Permeabilized NK cells (50×10⁶ cells per ml PBS), were injected into afive week old Balb/c mouse at three week intervals for a total of fourimmunizations. Three days after the last immunization, the immunizedmouse was sacrificed and splenocytes prepared using standard methods.Immune splenocytes were fused to the NS1 hybridoma cell line at a 1:1ratio using polyethylene glycol as described in Anderson, J. Immniol.143:1889 (1989). Following fusion, cells were cultured at limitingdilution in a 96-well plate in the presence of RPMI media containing 10%fetal calf serum and HAT selection medium. Individual supernatants werescreened for their reactivity with permeabilized and unpermeabilized NKcells, T cells, B cells, monocytes. Monoclonal antibody 5H10 (anti-PEN5)was selected as an antibody which reacted specifically with peripheralblood NK cells.

More specifically, the reactivity pattern of 5H10 was first determinedby testing purified peripheral blood lymphocytes obtained from healthyvolunteers using an Epics V flow cytometer (Coulter Electronics,Hialeah, Fla.). Peripheral blood lymphocytes (“PBLs”) purified asdescribed in the Methods and Materials, were stained by 2-color flowcytometry using rhodamine-conjugated anti-CD56 mAb, rhodamine-conjugatedanti-CD3 mAb, rhodamine-conjugated anti-CD20 mAb, FITC-conjugatedanti-CD16 mAb or biotinylated anti-PEN5 mAb in accordance with wellestablished techniques. The binding of biotinylated anti-PEN5 mAb wasrevealed using APC-conjugated avidin.

The results of the two color flow cytometry are shown in FIGS. 1Athrough 1D, in which the numbers in each quadrants indicate the percentof positive stained cells.

As shown in FIGS. 1A through 1D, the analysis of PBLs revealed a uniqueepitope, PEN5, to be expressed on the majority of CD56 ⁺ (FIG. 1A) andCD16 ⁺ (FIG. 1B) PBLs. In contrast, PEN5 was not significantly expressedon CD3 ⁺ T cells (FIG. 1C) or on CD20 ⁺ B cells (FIG. 1D).

To test cell surface expression of activated T cells and activated Bcells, peripheral blood T cells isolated as described were activatedwith optimal mitogenic concentrations of PHA and Con A. Splenic B cellswere activated with optimal concentrations of Staphylococcus aureasCowan strain I in accordance with standard laboratory protocols.Immunofluorescence staining was performed at days 2, 4, and 6 afteractivation. Neither T cell activation induced by mitogenicconcentrations of PHA or Con A (in the presence or absence of PMA), norB cell activation induced by Staphylococcus aureus Cowan strain I, for 1to 6 days induced the cell surface expression of the PEN5 epitope (SeeTable 1 below). Similarly, allogeneic T cell clones (CD3 ³⁰ CD4 ⁺ or CD3⁺ CD8 ⁺) did not express the PEN5 epitope (See Table 2).

Cell surface expression of the antigen recognized by the antibody 5H10on hematopoietic cells was also assessed by indirect immunofluorescenceand flow cytometry in accordance with established protocols. Assummarized in Table 1 below, cell surface staining of monocytes,granulocytes, platelets and erythrocytes also failed to reveal the PEN5epitope, confirming that PEN5 is an NK cell restricted molecule.

TABLE 1 Cell surface expression of PEN5 on hematopoietic cells. Celltype Relative Expression* Peripheral blood T cells − Activated T cells‡− Thymocytes − Peripheral blood NK cells ++ NK cell lines: YT.N17 − 3.3± NKL − Peripheral blood B cells − Splenic B cells − Activated Bcells^($) − Monocytes − Granulocytes − Platelets − Red blood cells −*The cell surface expression of 5H10 was assessed by indirectimmunofluorescence and flow cytometry; -:<5% positive stained cells;±:between 5 and 20% positive stained cells; ++:>60% positive stainedcells. ‡Peripheral blood T cells were activated with optimal mitogenicconcentrations of PHA and CON A, and immunofluorescence staining wasperformed at days 2, 4 and 6 after activation. ^($)Splenic B cells wereactivated with optimal mitogenic concentrations of Staphyllococcusaureus Cowan strain I, and immunofluorescence staining was performed atdays 2, 4 and 6 after activation.

TABLE 2 Absence of surface expression of PEN5 on cytotoxic T cell clonesCell surface expression* Clone CD3 CD2 CD4 CD8 CD56 PEN5 T4C1 + + + − −− 6.5 B4 + + + − − − 6.5 C1 + + + − − − 20.1 A2 + + + − − − 8.17 A + + +− + − 20.1 D8 + + − + − − T4T8C1 + + + − + − *The cell surface phenotypeof the indicated T cell clones was performed by immunofluorescence andflow cytometry. −:5% positive stained cells; +:>60% positive stainedcells.

To more precisely analyze the expression of PEN5 on NK cells, flowcytometric analysis of PEN5 expression was performed on freshly isolatedperipheral blood NK cells purified by negative selection usingimmunomagnetic bead depletion (see Materials and Methods). PEN5 wasbrightly expressed on 71.7±3.5% (mean±SEM, n=16) of these NK cellpreparations whose average phenotype was 75.6±3.3% CD56 ⁺, 4.2±4.0% CD16⁺ and 8.3±3.5% CD3 ⁺.

The phenotypic heterogeneity of peripheral blood NK cells required amore careful comparison of the relative expression of PEN5 and CD56. Thetwo-color flow cytometric comparison shown in FIGS. 1A through 1Dsuggested that PEN5 was preferentially expressed on the CD56 ^(dim)population. This was confirmed by comparing the expression of PEN5 onsorted populations of CD56dim and CD56bright NK cells, as shown in FIGS.2A through 2I.

Briefly, purified NK cells were sorted into CD56 ^(dim) and CD56^(bright) NK cell subsets using rhodamine-conjugated anti-CD56 mAb andflow cytometry. Unsorted NK cells, CD56 ^(dim) and CD56 ^(bright) NKcells were further analyzed for the expression of 5H10 usingbiotinylated anti-PEN5 mAb and FITC-conjugated avidin. Controls wereperformed using mouse isotype matched control IgM mAb. The results ofthis experiment are illustrated in FIGS. 2A through 2I, in which thenumbers in each histogram indicate the percentage of positively stainedcells. As shown in FIGS. 2A through 2I, PEN5 was expressed at a highdensity on 85.9 ±2.2% of CD56 ^(dim) NK cells (n=4), and at low densityon 31.1±5.3% of CD56 ^(bright) NK cells. These results indicate thathigh density cell surface expression of the PEN5 epitope is restrictedto the functionally differentiated CD56 ^(dim) NK cells. These resultsalso indicate that the cell surface expression of PEN5 defines twodistinct subsets of NK cells, PEN5 ⁺ and PEN5 ^(dim/−) which overlapwith the CD56 ^(dim) and CD56 ^(bright) NK cell subsets, respectively.

EXAMPLE 2

PEN5 expression is down-regulated by NK cell activation

CD56 ^(dim) and CD56 ^(bright) NK cells strongly differ in theirresponse to proliferative stimuli. Although CD56 ^(dim) NK cells do notproliferate in response to either IL-2 or the combination of ionomycinand PMA, CD56 ^(bright) NK cells proliferate in response to eitherstimulus. We took advantage of the recent observation that CD56 ^(dim)NK cells can be induced to proliferate in response to a combination ofLCM and ionomycin to correlate PEN5 expression with the NK cellproliferative state. Briefly, sorted CD56 ^(dim) and CD56 ^(bright) NKcells were activated for 20 days with ionomycin and LCM as described inthe Materials and Methods. At 0, 6, 8, 10, 14, and 20 days of culture,aliquots of the activated NK cell populations were analyzed for theircell surface phenotype by flow cytometry using isotype matched controlmAb, anti-CD56 and 5H10 mAb. The results illustrated in FIG. 3 indicatethe percent of positively stained cells (%); the total mean fluorescenceintensity is indicated below in the histograms.

As shown in FIG. 3, activation of CD56 ^(dim) NK cells resulted in thetemporal reduction of PEN5 expression. In parallel, the cell surfaceexpression of CD56 was temporally increased, and after 20 days ofactivation, the cell surface expression of PEN5 and CD56 on the CD56^(dim) NK cells was similar to that of unactivated CD56 ^(bright) NKcells (i.e: PEN5 ^(dim/−) and CD56 ^(bright)). These results areconsistent with the absence of PEN5 from the cell surface of long termhuman NK cell clones (A. Moretta, personal commuication). In addition,PEN5 was not expressed on leukemic NK cells (CD3:TCR⁻, CD16 ⁺, CD56 ⁺)isolated from patients with granular lymphocyte proliferative disorder(See, FIG. 4). Finally, 5H10 was absent or dimly expressed on three longterm human NK cell lines, 3.3, NKL and YT.N17. These results indicatethat PEN5 expression inversely correlates with the NK cell proliferativecapacity.

EXAMPLE 3

Biochemical characterization of the PEN5 epitope. 5H10Immunoprecipitates Two Polydispersed Bands

Radioiodinated lysates prepared from resting NK cells wereimmunoprecipitated using the 5H10 (anti-PEN5) mAb or an isotype matchedmouse IgM control mAb. Immunoprecipitates were then separated undernon-reducing conditions on SDS-polyacrylamide gels (6% SDS). The resultsare shown in FIG. 5.

As illustrated in Figures, two diffuse bands were selectivelyimmunoprecipitated by the 5H10 mAb. The average molecular weight (m.w.)of the larger species, PEN5α, was 227±4 kDa (n=12). The m.w. range ofthe polydispersed PEN5 species was 210±3 kDa to 245±5 kDa. The averagem.w. of the smaller species, PEN5β was 140±3 kDa, with a range of 123±3kDa to 170 ±4 kDa. The migration of both PEN5α and β molecules aspolydispersed bands suggested that they were highly glycosylated.

PEN5 α and β Are Carbohydrates With Keratanase I-Sensitive Chains

These results were confirmed in deglycosylation experiments, the resultsof which are shown in FIG. 6. In these experiments detergent lysatesprepared from radioiodinated NK cells were immunoprecipitated using 5H10mAb. Affinity-purified PEN5α and β glycoproteins were eluted from theantibody-coated sepharose beads using 0.15M NH₄OH, pH 10.5. Aliquots ofthis dried sample were then subjected to deglycosylation for 24 hr at37° C. using PNgase F (lane 6), O-glycanase (lane 3), keratanase I (lane2), O-glycanase and keratanase (lane 4), neuraminidase (lane 6), andPNgase F and neuraminidase (lane 7). Control eluates incubated in PBSwithout any enzymes were separated in lane 1. Samples were separatedunder non-reducing conditions on a 6-12% SDS-polyacrylamide gradientgel.

Compared to the migration of untreated PEN5 glycoproteins (FIG. 6, lane1), PNgase F treatment induced the disappearance of PEN5α from the210-245 kDa m.w. range, and the appearance of a deglycosylated form ofPEN5α migrating at 20-25 kDa (c2). In contrast, the apparent mobility ofPEN5β was reduced by only ˜20 kDa after PNgase F incubation. Treatmentof PEN5 glycoproteins with O-glycanase (FIG. 6, lane 3) did notsignificantly affect their SDS-PAGE migration pattern. These resultsindicate that the PEN5α and PEN5β differ markedly in their carbohydratescomposition, and that ˜85% of the apparent m.w. of PEN5α is due toN-linked carbohydrates.

PEN5α Contains 80% N-Linked Kertanasse-Sensitive Carbohydrates WhereasPEN5β Contains 80% O-Linked Keratanase-Sensitive Carbohydrates

The extensive N-linked glycosylation of PEN5α suggested that it might bea member of one of the two major groups of glycoproteins characterizedby such high carbohydrate content (50 to 90%), i.e: proteoglycans andmucin-type glycoproteins. Chondroitinase ABC, heparitinase andheparinase did not affect the migration pattern of PEN5α or PEN5β (datanot shown). By contrast, incubation of PEN5 molecules with keratanase Ireduced the apparent m.w. of PEN5α from 210-245 kDa to 35-40 kDa (FIG.6, lane 2; c1). It is likely that the difference between the PNgaseF-digested (35-40 kDa) c1 core protein (FIG. 6, lane 6) and thekeratanase-digested (25-30 kDa) c2 core protein (FIG. 6, lane 4), is theconsequence of a more complete deglycosylation of the PEN5αglycoprotein. Whereas keratanase treatment only slightly reduced thepolydispersity of PEN5β, the combination of O-glycanase and keratanase Itreatment reduced the apparent m.w. of PEN5α from 120-170 kDa to 25-30kDa (FIG. 6, lane 4). Taken together, these results indicate that, byweight, PEN5α contains ˜80% N-linked keratanase I-sensitivecarbohydrates, whereas PEN5β contains ˜80% O-linked keratanaseI-sensitive carbohydrates. In addition, treatment with neuraminidaseinduced a slight reduction in the polydispersity, as well as a shift inthe apparent m.w. of both PEN5α and β, indicating that sialic acidresidues are also present on both glycoproteins (FIG. 6, lane 5).Treatment of PEN5 glycoproteins with a combination of PNGase F andneuraminidase (FIG. 6, lane 7). resulted in the same effect that PNGaseF alone, confirming the presence of terminal sialic acid residues onN-linked carbohydrates present on PEN5α. The c1 and c2 deglycosylatedforms of PEN5α and β proteins were not immunoprecipitable by the 5H10mAb (data not shown), indicating that the epitope recognized by theanti-PEN5 mAb requires the keratanase I-sensitive carbohydrate chains.

EXAMPLE 4

Reactivity of anti-PEN5 mAb with keratan sulfate glycosaminoglycans.

In order to test whether the anti-PEN5 mAb was directed against keratansulfate carbohydrates, we next examined the effect of exogenous keratansulfate carbohydrates on the binding of PEN5 mAb to NK cells.Radioiodinated 5H10 (anti-PEN5) mAb was combined with variousconcentrations of bovine cornea keratan sulfate proteoglycan (BC), andthe mixture was then incubated with NK cells.

Briefly, ¹²⁵ -labeled 5H10 mAb (1×10⁶ cpm/sample) was preincubated for20 min at 4° C. in PBS in the presence of the concentrations of bovinecornea keratan sulfate (BC) indicated in FIG. 7A. The mixture was thenadded to NK cells for another 20 min incubation at 4° C., prior to threewashes in PBS-1% BSA. Samples were counted in a τ-counter, and resultswere expressed as mean cpm of duplicate samples (SD<10%). When used inincubation with NK cells or anti-PEN5 mab, the following carbohydratesused at 10 mg/ml were without any effect on 5H10 binding to NK cellsurface: chondroitin sulfate B, heparin, heparan sulfate, dextransulfate, GlcNAc, mannose 6-phosphate, lactose, galactose-6-phosphate,fucose, glucose 6-phosphate, glucose and galactose.

As shown in FIG. 7A, the binding of radiolabeled 5H10 mAb to NK cellswas inhibited in a dose-dependent manner in the presence of BCproteoglycan. Preincubation of NK cells with the same concentrations ofBC proteoglycan did not affect the binding of 5H10 mAb (data not shown),indicating that the anti-PEN5 mAb reacted with carbohydrate determinantspresent on keratan sulfate glycosaminoglycans. Incubation of anti-PEN5mAb with simple sugars or other glycosaminoglycans was without anyeffect (see Brief Description of FIG. 7A).

Furthermore, treatment of NK cells with keratanase I induced a 58.5%±8.4(n=4) decrease in the reactivity of 5H10 mAb with NK cells (FIG. 7B). InFIG. 7B, peripheral blood NK cells were incubated in PBS-1% BSA for 3 hror 45 min at 37° C. with glycosidases (0.025 U/ml) or proteases (5mg/ml) respectively. Cell surface expression of PEN5 epitope was thenanalyzed by flow cytometry using 5H10 mAb. Percent modulation wascalculated as the ratio of the total linear mean fluorescence intensityof the treated cells over that of untreated control cells. Asillustrated in FIG. 7B, parallel treatment of NK cells withchondroitinase ABC or neuraminidase did not have any effect on 5H10reactivity. Interestingly, the 5H10 epitope was totally insensitive totrypsin and chymotrypsin but was removed by proteinase E treatment.

Finally, an ELISA was used to compare the binding of 5H10 (anti-PEN5)and 5D4 (anti-keratan sulfate) to the keratan sulfate proteoglycansexpressed in various tissues. The antigenicity of 5H10 mAb for aggrecanproteoglycans was analyzed by ELISA as described in Materials andMethods. The anti-keratan sulfate mAb 5D4 was used as a positivecontrol. Chondroitinase ABC was used at 0.04 U/ml, keratanase I was usedat 0.05 U/ml and keratanase II was used at 0.004 U/ml, for 1 hr at 37°C.

As illustrated in FIGS. 7C through 7E, the 5H10 mAb (cross-hatched)recognized aggrecan-type proteoglycans derived from embryonic chickcartilage (CD1, FIG. 7C) and from bovine nasal to cartilage (BNC, FIG.7D). As a positive control, the anti-keratan sulfate mAb 5D4 (open bars)also reacted with untreated CD1 and BNC, whereas its reactivity withkeratanase-treated samples was reduced. Treatment of CD1 and BNC witheither keratanase I or II, reduced 5H10 reactivity. Treatment of CD1 andBNC with chondroitinase ABC is known to increase the expression ofkeratan sulfate epitopes. Consequently, digestion of CD1 and BNC withchondroitinase ABC increased the binding of both 5H10 and 5D4. As anegative control, neither mAb recognized the Swarm rat chondrosarcomaaggrecan (RC), which does not contain keratan sulfate (FIG. 7E).Although 5D4 also reacted with the keratan sulfate proteoglycan isolatedfrom shark cranial cartilage (SHK), 5H10 did not. These results indicatethat the 5H10 epitope is present in some, but not all, keratan sulfatechains. Although 5H10 can clearly recognize an epitope expressed oncertain keratan sulfate chains, the epitope expressed on the PEN5molecule on NK cells is not simply a keratan sulfate chain since flowcytometric analysis using 6 distinct anti-keratan sulfate mAbs 1B4, 2D3,3D2, 4D1, 8C2, and 5D4 did not detect binding to NK cells (data notshown).

Taken together, these results indicate that 5H10 recognizes an epitopethat, although present on keratan sulfate carbohydrates, is distinctfrom the standard sulfated polylactosamine repeat sequence, Ga1b1-4(sulfated)GlcNAc.

EXAMPLE 5

PEN5 glycoproteins are expressed at the NK cell surface as extendedrod-like structures.

Transmission electron microscopy was performed on NK cells stained byindirect immunofluorescence using the 5H10 mAb and a gold-labeledanti-IgM developing reagent. Ultrathin sections of NK cells showedextensive labeling at the cell surface (see, FIG. 8A -8C). Labeling wasgenerally continuous around the entire cell profile, although in somecell preparations, there was relatively more labeling over microvilli.More striking was the distance between the plasma membrane and the goldlabel, which averaged 43.4 ±12.8 nm (n=50). This result suggests that,like other cell surface mucins, the membrane-bound glycoproteinscarrying the PEN5 epitope are extended thread-like proteins.

Taken together, our results indicate that the PEN5 epitope is in part acarbohydrate determinant that can be expressed on keratan sulfatechains. First, keratan sulfate glycosaminoglycans selectively competewith PEN5 molecules for binding to the 5H10 (anti-PEN5) mAb. Second,treatment of NK cells with keratanase I down-regulates the cell surfaceexpression of the PEN5 epitope. Third, the 5H10 mAb recognizes twodistinct aggrecan-type keratan sulfate proteoglycans. Keratan sulfatesare glycosaminoglycans consisting of repeated Ga1b1-4 (sulfated)GlcNacdisaccharides. Within this constraint, differential branching of thedisaccharide subunits, differential sulfation of GlcNAc, anddifferential fucoslyation and/or sialylation of the Ga1b1-4(sulfacted)GlcNAc can lead to heterogeneity in individual keratansulfate chains. The lack of reactivity of anti-PEN5 mAb with thekeratan-sulfate proteoglycan SHK isolated from shark cranial cartilagesuggests that the standard lactosaminoglycan repeat sequence is not theepitope recognized by 5H10. Rather, our data indicate that 5H10recognizes an unusual sulfated polylactosamine epitope present on somebut not all keratan sulfate glycosaminoglycans.

EXAMPLE 6

Identification of Tissue-Infiltrating Natural Killer Cells Expressingthe Mucinlike Glycoprotein PEN51

Materials and Methods

The following methods and materials apply to Examples 6A through 6D.

Source of Tissues. Histologically normal fetal (20 week gestation) andadult human tissues were obtained from surgical and autopsy specimens.Frozen tissues embedded in OCT compound (Baxter Corp., McGaw Park, Ill.)were stored at −70° C. until needed. All tissues were used as frozentissue sections and were adequately preserved histologically. The panelof normal tissues that were screened included adrenal, brain, breast,cervix, colon, esophagus, heart, kidney, liver, lung, lymph node, ovary,peripheral nerve, pancreas, skeletal muscle, skin, small intestine,spleen, stomach, testis, thyroid, tonsils, thymus, and uterus.

Reagents. Anti-5H10 was used at a dilution of 1:400 (2.5 mg/ml) inphosphate buffered saline (PBS) containing 0.06% crystalline bovineserum albumin (BSA) and 0.1% sodium azide. Purified mouse IgM (CoulterImmuology, Hialeah, Fla.) served as the negative control. For use it wasdiluted to the same concentration, with the same buffer solution as thetest antibody. N901, a murine monoclonal antibody of the IgG1 subclass,binds to the NKH1 antigen (CD56) expressed on NK cells. The antibody wasused at a dilution of 1:664 (2.5 mg/ml) in PBS containing 0.06%crystalline bovine serum albumin (BSA) and 0.1% sodium azide.Biotinylated affinity purified goat anti-mouse IgM (m chain specific)and horse anti-mouse IgG (heavy+light chain specific) antibodies (VectorLaboratories, Inc,. Burlingame, Calif.) were utilized as secondaryantibodies at a dilution of 1:150 in PBS containing 2% human AB⁺ serumand 0.1% sodium azide. Avidin-biotin-peroxidase complexes (Vector) wereused as the labeling reagent at a dilution of 1:1:80 in PBS.

Immunohistochemistry. Immunohistochemical studies were performed usingthe avidin-biotin immunoperoxidase technique [Rice, et al., Am. J.Path., 138:385, (1991)]. To assure that tissue sections adhered, slideswere coated with poly-L-lysine (Sigma Chemical Co., St. Louis, Mo.)reconstituted in purified water. Frozen sections were cryostat cut (6-8mm thick), collected onto coated slides, air dried and fixed in 2%neutral buffered paraformaldehyde at 4° C. for 20 minutes, followed byseveral washes with PBS. To block endogenous biotin content, and reducecross-reactivity of the biotinylated antibody, all tissues wereincubated with a solution of avidin (vector) and 10% normal horse serum(Vector) in BSA dilution buffer, at room temperature for 15 minutes.Tissue sections were drained of avidin/horse serum buffer and incubatedwith the antibody at 4° C., overnight. After washing in PBS, slides wereincubated for 30 minutes in 0.3% hydrogen peroxide and biotin blockingsolution to quench endogenous peroxidase activity and to block remainingavidin. Sections were then washed with PBS, incubated with eitherbiotinylated goat anti-mouse IgM or horse anti-mouse IgG antibodies for30 minutes, washed in PBS, incubated with avidin-biotin-peroxidasecomplexes for 45 minutes, and then washed again with PBS. Afterincubating the slides for 5 minutes in Tris-Imidazole/HCL buffer, theperoxidase reaction was initiated by incubating for 5 minutes with3,3-diaminobenzidine (DAB) (Sigma Chemical Co.) dissolved inTris-Imidazole/HCL buffer containing 0.11% hydrogen peroxide. Tissuesections were washed in water, counterstained with Harris hematoxylin,and dehydrated through graded alcohols and xylenes. Coverslips were thenmounted on slides with E-Z-Mount mounting media (Shandon Inc.,Pittsburgh, Pa.).

Transmission Electron Microscopy. Peripheral blood NK cells purified asreported in Vivier, et al., J. Immunol., 146:206, (1991) were firststained using 5H10 (anti-PEN5) and colloidal gold-labeled goatanti-mouse IgM (Amersham). After fixation for 1 hour in 0.1%glutaraldehyde, 2% paraformaldehyde, the stained cells were examined bytransmission electron microscopy as described in Watkins, et al.,Carbohydrate Res., 213:185, (1991).

EXAMPLE 6A

Comparative expression of PEN5 ⁺ and CD56 ⁺ lymphocytes infiltratinglymphoid tissues.

Although CD56 is expressed on the surface of most peripheral blood NKcells, its density of expression on most NK cells is quite low. Becauseof this, antibodies reactive with CD56 may not be ideal reagents for theidentification of tissue infiltrating NK cells. The relative inabilityof anti-CD56 to detect NK cells in lymphoid tissues is demonstrated inFIGS. 9A through 9H and FIGS. 10A through 10B in which CD56 ⁺ cells arerarely detected in lymph node, tonsil, or thymus. In contrast,antibodies reactive with PEN5 identified lymphocytes infiltrating eachof these tissues. Whereas PEN5 ⁺ cells were scattered throughout thelymph node, they tended to be concentrated in the parafollicular areasof the tonsil. At higher magnification, PEN5 ⁺ cells were observed to beround or oval or occasionally elongated. They were generally larger thanresting tissue lymphocytes, containing a relatively abundant cytoplasm.The nuclei were set eccentrically within the cells, and were slightlylarger than those of resting lymphocytes. The nuclear chromatin wasdense and homogeneous. Immunostaining was usually in the region of theplasma membrane, but was also seen in the cytoplasm.

EXAMPLE 6B

Comparative expression of PEN5 ⁺ and CD56 ⁺ lymphocytes in fetal andadult tissues.

Because fetal liver and fetal thymus have been implicated as sites of NKcell differentiation, we compared the expression of PEN5 ⁺ and CD56 ⁺lymphocytes in each of these tissues to that of their adultcounterparts. As shown in FIG. 10, CD56 ⁺ cells were not easily detectedin either fetal or adult thymus. In each of these tissues, scatteredlymphocytes expressing low levels of CD56 could be detected at highmagnification, suggesting that CD56 ⁺ cells are present, but difficultto detect using this histochemical method. This might result fromliability of the antigen under these fixation conditions, or the lowlevel of CD56 expression, since Sanchez, et al [J. Exp. Med. 178:1857(1993)] have shown that CD56 ⁺ lymphocytes can be identified in thesetissues using flow cytometric analysis. In contrast, PEN5 ⁺ cells wereeasily detected, scattered throughout both adult and fetal thymus. Thedensity of PEN5 ⁺ cells was consistently greater in fetal thymus than inadult thymus. Occasional CD56 ⁺ cells could be detected in adult liver,but again, the intensity of staining was very weak (FIGS. 11A through11H). Scattered PEN5+ cells were easily detected in the adult liver, dueto their more intense staining. Relatively more PEN5 ⁺ cells wereobserved in fetal liver compared to adult liver. At highermagnification, PEN5 expression in liver infiltrating lymphocytesappeared, at least in part, cytoplasmic. Previous results have shownthat mucin-like glycoproteins can be identified in the trans-Golgireticulum and in cytoplasmic vesicles that eventually fuse with theplasma membrane [Watkins, et al., Carbohydrate Res. 213:185 (1991)]. Itis possible that liver infiltrating NK cells express PEN5 primarily inthese intracellular compartments.

The above results demonstrate PEN5 ⁺ lymphocytes were particularlyprevalent in fetal liver and fetal thymus. Recent studies suggest thatNK cells and T cells arise from a common bone marrow-derived progenitorcell [Sanchez, et al., J. Exp. Med. 178:1857 (1993); Lanier, et al.,Immunol. Today 13:392 (1992); Rodewald, et al., Cell 69:139-(1992); andKoyasu, et al., J. Exa. Med. 179:1957 (1994)]. Homing of these cells tothe fetal liver, a major site of prenatal hematopoiesis, fosters thedevelopment of CD56 ⁺ cells that resemble peripheral NK cells in bothphenotype and function. Some evidence suggests that these cells candifferentiate into T cells if they leave the liver and home to thethymus [Sanchez, et al., J. Exp. Med. 178:1857 (1993]. In the absence ofthe thymic microenvironment, these cells can differentiate into NK cellsif provided with appropriate growth factors [Sanchez, et al., J. Exp.Med. 178:1857 (1993); and Koyasu, et al., J. Exp. Med. 179:1957 (1994)].Similarly, several studies have shown that in vitro culture of immaturethymocytes in the presence of IL-2 results-in the differentiation ofcells which phenotypically and functionally resemble peripheral blood NKcells [Sanchez, et al., J. Exp. Med. 178:1857 (1993); Koyasu, et al., J.Exp. Med. 179:1957 (1994); Michon, et al., J. Immun. 140:3660 (1988);and Mingari, et al., J. Exp. Med. 174:21 (1991)]. Some of these studiesrely on the characterization of lymphocyte clones that grow out ofselected fetal and adult tissues. As clonal selection may impart a biason any analysis of cell populations, the observation that PEN5 ⁺lymphocytes are present in fetal liver and thymus provides unbiasedevidence for the differentiation of NK cells in these tissues. Althoughrelatively few CD56 ⁺ cells were identified at these sites usinghistochemical analysis, this result might reflect the low density ofexpression of this NK marker. CD56 ⁺ lymphocytes have been detected inboth fetal liver and fetal thymus using flow cytometric analysis[Sanchez, et al., J. Exp. Med. 178:1857 (1993)]. Our results suggestthat PEN5 expression can be expected to be a more sensitive marker oftissue infiltrating NK cells than CD56 expression.

EXAMPLE 6C

Expression of PEN5 antigen on non-lymphoid cells.

Antibodies reactive with PEN5 also recognized some non-leukocytic cells.These were generally epithelial cells found in the esophagus, cervix,endometrium, trachea, bile ducts, colon and pancreas. The most dramaticexample of this non-lymphoid staining was seen in the lung and colon,where anti-PEN5 strongly stained the mucous layer lining bronchial andcolonic epithelial cells (FIGS. 12A through 12F). The specificity ofthis staining was confirmed by the inability of either isotype matchedcontrol antibody or anti-CD56 to stain epithelial mucosa.

EXAMPLE 6D

Co-expression of PEN5 and TIA-1 in tissue infiltrating lymphocytes.

Further evidence that PEN5 ⁺ tissue infiltrating lymphocytes are NKcells comes from double labeling experiments using a monoclonal antibodyreactive with TIA-1 (2G9, IgG1), a cytotoxic lymphocyte-restrictedgranule protein [Anderson, et al., J. Immunol. 144:574 (1990); Tian, etal., Cell 67:629 (1991); and Sale, et al., Arch. Path. Lab. Med. 116:622(1992)]. In these experiments, PEN5 ⁺ cells were identified in spleenand appendiceal lymphoid tissue using FITC-tagged anti-5H10. These samesections were also labeled using phycoerythrin-tagged anti-2G9. As shownin FIGS. 13A through 13D, all four PEN5 ⁺ lymphocytes scatteredthroughout the spleen were also TIA-1 ⁺. Consistent with thelocalization of these antigens, PEN5 staining is largely confined to thecell surface, whereas TIA-1 staining is cytoplasmic, and granular. SomePEN5-cells expressed TIA-1. These cells are likely to be cytotoxic Tcells which express TIA-1, but not PEN5. In the appendix, only one outof four PEN5 ⁺ lymphocytes co-expressed TIA-1. This result suggests thatin some tissues, PEN5 might identify less differentiated NK cells thatdo not possess defined cytotoxic granules. Alternatively, these resultsmight reflect changes in the expression of TIA-1 that are related to NKcell differentiation. Table III tabulates the percentage of PEN5 ⁺tissue infiltrating lymphocytes expressing TIA-1 in several tissues. Assummarized below, whereas the majority of PEN5 ⁺ lymphocytes co-expressTIA-1 in spleen and liver, this is not the case in tonsil or appendix,where most PEN5 ⁺ lymphocytes do not express TIA-1. Whether these tissuespecific differences reflect different stages of NK celldifferentiation, or different types of tissue infiltrating lymphocyteremains to be elucidated.

TABLE III Expression of TIA-1 in PEN5 ± Tissue Infiltrating LymphocytesTISSUE DONOR# % TIA-1⁺ Spleen 1 100 2  64 3  84 4 100 5  88 Average: 87 + 13 Tonsil 1  16 2  40 3  36 4  28 Average:  30 + 9 Liver 1  96 2 88 3 100 4  84 5  92 Average:  92 + 6 Appendix 1  12 2  0 3  12 4  4Average:  7 + 5 ⁺Dual labeling of the indicated tissues was performed asdescribed in the Materials and Methods to Example 6, and in the briefdescription to FIGS. 13A through 13D. The percentage of PEN5⁺ cells thatexpressed TIA-1 is indicated. Tissues from 4 or 5 independent donorswere evaluated, and the mean standard error is reported.

Taken together, the results provided in Examples 6A through 6Dillustrate a number of important findings. We have used a monoclonalantibody reactive with a sulfated poly-N-lactosamine epitope expressedon the NK cell restricted glycoprotein PEN5 to survey the presence oftissue-infiltrating NK cells in lymphoid and non-lymphoid tissues.Whereas antibodies reactive with CD56 were unable to efficiently detectall tissue infiltrating NK cells, PEN5 ⁺ lymphocytes were readilyidentified in multiple tissues. Assuming that PEN5 is expressedsimilarly on both tissue infiltrating and circulating lymphoid cells,these results suggest that NK cells can infiltrate multiple lymphoid andnon-lymphoid tissues to mediate their immune functions. In theperiphery, PEN5 is selectively expressed on large granular lymphocytespossessing cytotoxic effector function. These cells express low levelsof CD56, which might account for the inability of antibodies reactivewith CD56 to recognize these cells in tissues. Double staining with thecytotoxic granule marker, TIA-1, supports the conclusion that PEN5 ⁺lymphocytes infiltrating some tissues (e.g. spleen and liver) containcytotoxic granules. surprisingly, however, many PEN5 ⁺ cellsinfiltrating other tissues (e.g. tonsil and appendix) did not co-expressTIA-1. This result suggests that in some tissues, PEN5 might beexpressed on agranular lymphocytes.

The PEN5 epitope recognized by monoclonal antibody 5H10 is related tokeratan sulfate, which is itself a member of the polylactosamine familyof sugars. The two isoforms of PEN5 thus resemble a keratan sulfateproteoglycan (PEN5β) and a keratan sulfated mucin (PEN5α). Secretedmucins derivatized with keratan sulfate have been identified in thetracheal mucosa [Kim, et al., Exp. Lung Res. 17:533 (1991)]. It ispossible that the recognition of the tracheal and gastrointestinal mucinlayer by anti-5H10 results from its recognition of these keratansulfated mucins. We have previously shown that anti-5H10 can recognizekeratan sulfate-bearing proteoglycans derived from several tissues,including embryonic chick cartilage and bovine nasal aggrecan [Vivier,et al., J. Exp. Med. 178:2023 (1993)]. In epithelial cells, mucins aresecreted to provide protection against environmental toxins [Strous, etal., Critical Rev. in Biochem. and Molec. Biol. 27:57 (1992)]. It ispossible, by analogy, that PEN5 is expressed on differentiated largegranular NK cells to protect them against their own cytotoxic effectormolecules. The extended, rod-like structure of PEN5 demonstrated bytransmission electron microscopy could facilitate such a functionalrole. Cell surface mucins have also been identified as ligands forlymphocyte adhesion molecules involved in tissue homing [Lasky, et al.,Cell 69:927 (1992)]. It is therefore possible that the expression ofPEN5 on terminally differentiated NK cells allows its subsequentinfiltration into the various tissues in which these cells are found.

What is claimed is:
 1. A partially purified preparation of PEN5.
 2. Apartially purified preparation of enzymatically deglycosylated PEN5. 3.A partially purified preparation of PEN5α.
 4. A partially purifiedpreparation of enzymatically deglycosylated PEN5α.
 5. A partiallypurified preparation of PEN5β.
 6. A partially purified preparation ofenzymatically deglycosylated PEN5β.